Plant Physiol. (1971) 47, 98-103

Induction of Cold Acclimation in Cornus stolonifera Michx.'12Received for publication June 29, 1970

L. H. FUCHIGAMI,3 C. J. WEISER, AND D. R. EVERT4Laboratory of Plant Hardiness, Department of Horticultural Science, University of Minnesota, St. Paul, Minne-sota 55101

ABSTRACT

A warm (20 to 15 Celsius day or night) preconditioningtreatment enhanced cold acclimation of Cornus stoloniferabark under short-day conditions when plants were precon-

ditioned for at least 4 weeks. Warm preconditioning in-hibited the acclimation of plants subjected to long photo-

periods. Removing leaves from plants exposed to low tem-

peratures and short days inhibited acclimation. Removal ofbuds did not affect acclimation. Plants did not acclimateunless they were exposed to at least 4 weeks of short photo-periods prior to defoliation. Plants began to acclimate to

cold at the time of growth cessation but not before. Whenhalf of the leaves were removed from plants, the defoliatedand foliated branches both acclimated as well as brancheson completely foliated plants. Girdling the phloem betweenfoliated and defoliated branches prevented acclimation ofthe latter regardless of the position of the girdle in relationto the root system and the defoliated branch. When all ofthe leaves of plants were covered with aluminum foil to ex-

clude light after 0 or 4 weeks of exposure to short days, theresults resembled a defoliation study, i.e., plants withleaves covered at the start of thie experiment failed to ac-

climate, and those covered after 4 weeks acclimated to some

extent but less than uncovered control plants. Under long-day conditions plants with all leaves covered failed to ac-

climate, and plants with none or half of their leaves coveredacclimated equally and to a limited extent. Under short-dayconditions, however, the covered branches of partiallycovered plants acclimated more than th-eir uncoveredcounterparts or branches of totally uncovered plants.

In nature the living bark of many woody plant species ac-climates to cold in two stages during the autumn (5, 7). Theresults of controlled environment studies have indicated thatthe first stage of acclimation is induced by short days (7, 8, 18)and the second stage by low temperatures (5, 18). Russianworkers have hypothesized that there is a third stage or

type of acclimation which is induced by prolonged exposure tovery low temperatures in midwinter (15).

Results of controlled environment studies have also suggestedthat endogenous rhythms influence cold acclimation. During

I This research was supported in part by a grant from the Louis W.and Maud Hill Family Foundation.

2 Scientific Journal Series Paper No. 7265 of the Minnesota Agricul-tural Experiment Station.

3Present Address: Department of Horticulture, Oregon State Uni-versity, Corvallis, Oregon 97331.

4Present Address: Department of Plant and Soil Science, Universityof Vermont, Burlington, Vermont 05401.

the spring growth-flush, Cornus stolonifera plants cannot be in-duced to acclimate regardless of environmental conditions (3,18). Conversely, apple trees grown in a warm greenhouse underartificially long photoperiods in the autumn acclimate to someextent in spite of the supposedly noninductive environment (5).

Recent studies of the first stage of acclimation in several de-ciduous woody species have revealed that leaves exposed toshort days are the source of a translocatable hardiness-promotingfactor(s) (5, 8), and that leaves exposed to long days are asource of factors which inhibit hardiness (5, 8). Removal ofleaves from short-day induced plants inhibits bark acclimation,whereas removal of leaves exposed to long days has the oppositeeffect (5, 8). Grafting studies with diverse climatic races of C.stolonifera have demonstrated that the hardiness-promotingfactor(s) from the leaves of one genotype can enhance coldacclimation in the bark tissues of another genotype (3). Irvingand Lanphear (8) have reported that the hardiness-promotingfactor(s) is translocated from leaves to overwintering tissues viathe xylem.

Studies in which different parts of the same plant are exposedto different environments have shown that the low temperature-induced phase of cold acclimation does not involve a translocatedfactor(s) (5). In spite of this basic difference, either low tem-perature or short days can induce acclimation in the absence ofthe other inductive factor. Plants exposed to low temperaturesand long days will, in time, acclimate fully. Similarly, exposureof plants to short days and relatively high temperatures results insubstantial, although not full, hardiness (18).Even though these studies have contributed considerably to

our knowledge of the first stage of acclimation, there are stilla number of unresolved questions. For example, the influence ofleaf factors on bark acclimation has been characterized, butlittle is known about the possible influence of root, bud, or stemfactors. Buds have been shown to be the photosensitive perceptorsites which are involved in the breaking of dormancy in Fagussylvatica L. (10), and leaves or buds are known to be the site ofperception of light for the onset of physiological rest in manyplant species (2, 9, 19, 20). The translocation path of the hardi-ness-promoting factor(s) from short-day induced leaves to theoverwintering tissues has not been studied thoroughly. The rolesof temperature and photoperiod have been studied during theacclimation process (6-8, 18), but we know little about how thedevelopmental status or the previous environmental history ofthe plant may affect its ability to acclimate.

This study was designed to examine the possible role of bud,root, and stem factors in cold acclimation and the translocationroute of hardiness-promoting factor(s) and to assess the influenceof environmental preconditioning on the first stage of cold ac-climation.

MATERIALS AND METHODS

Two clonally propagated northern races of C. stoloniferaMichx. native to Minnesota and North Dakota were used in the12 studies (13). All plants in each experiment were of a single

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INDUCTION OF COLD ACCLIMATION

clone. Prior to study plants were actively growing in a warm(24C) greenhouse at a 16-hr photoperiod. In growth chamberstudies, the light was supplied by a mixture of cool white fluores-cent and incandescent bulbs. The intensity at pot height was8.2 X 101 ergs/cm' sec at 15 C as measured by a YSI Ketteringmodel 65 radiometer. Short-day treatments consisted of naturalautumn photoperiods in field studies and either 10- or 12-hrphotoperiods in the growth chamber studies. Either the 10- or12-hr photoperiod causes growth cessation and induction of restperiod in both of the clones studied. The long-day treatments inboth field and growth chambers consisted of the short photo-period plus a 3- to 4-hr interruption of the middle of the darkperiod with light from incandescent bulbs.

Unless otherwise indicated, day-night temperature regimes ingrowth chamber studies consisted of a 20 to 15 C preconditioningperiod followed by 15 to 5 C for inducing acclimation. All of thetreatments and hardiness tests were run in triplicate. To testhardiness, stems (internodes) were cut into 2-cm-long internodesegments and were subjected to a controlled freezing test aspreviously described (3). Internode segments were frozen inDewar flasks at a rate of 10 C per hour. Flasks were removedfrom the freezer at 2-C intervals; the internode segments wererewarmed and incubated in a humid chamber at room temper-ature. After 7 days the samples were scored for survival. Anysegments not showing discoloration and breakdown of cells inthe cambium or bark were rated as living. Experience has shownthat living samples are capable of forming callus when incubatedfurther for 20 to 30 days. Freezing tests were run in triplicate,and in these studies there was almost always perfect agreementin the results of survival scoring on all three segments from eachtreatment at each test temperature. In rare instances when allsamples did not agree, the atypical sample was disregarded.Hardiness is expressed in figures as the lowest temperature atwhich samples were uninjured (lowest survival temperature).Growth was evaluated by periodically measuring stem lengthand by averaging the measurements. Differences in final growthbetween treatment means were evaluated by Duncan's multiplerange test (14).

RESULTS

For clarity the 12 studies conducted are referred to by numberin the ensuing discussion. In the interest of brevity, data arepresented in Figures for only six of the 12 studies conducted, andthe results of the other six supporting studies are discussed briefly.

Study 1 indicated that low preconditioning temperatures notonly failed to enhance acclimation but, in fact, inhibited theprocess. Three plants were subjected to short days and a low day-night temperature regime of 15 to 5 C continuously for 40 days.Another lot of three plants was subjected to higher day-nighttemperatures (20 to 15 C) for 28 days before they were trans-ferred to 15 to 5 C for the balance (12 days) of the 40-day experi-mental period. Plants given the warm preconditioning treatmentgrew more but also became more cold resistant (-16 C) thanthose exposed continuously to low temperatures for 40 days(-10 C) (Fig. 1).

In study 2 the influence of the duration of a warm precondi-tioning treatment (20 to 15 C) was examined. Ten lots of threeplants each were subjected to either 0, 1, 2, 3, or 4 weeks of pre-conditioning at either long or short photoperiods before theywere transferred to short days and a 4-C constant temperaturefor an additional 17, 16, 15, 14, or 13 weeks, respectively. Thehardiness of plants in the 10 treatments was compared at the endof 17 weeks. Figure 2 shows the results.As in the previous study, a warm preconditioning period (20

to 15 C) for 4 weeks resulted in effective acclimation (to -26 C)under short-day conditions. When there was less than 4 weeks ofpreconditioning, acclimation was not enhanced; in fact, no pre-

E2

4Z A

0

04

4

LOWESTSURVIVALTEMPERATURE(DAY 40)

-16,A

-10, a

TIME (cOaysI

FIG. 1. The influence of preconditioning temperatures on the elonga-tion and hardiness of C. stolonifera stems. The hardiness (lowest sur-vival temperature) was determined on the 40th day of the experiment.The letters A and B indicate that the average stem elongation was sig-nificantly different at the 0.05 level of significance as determined by theDuncan multiple range test. Temperature regimes were as shown.

conditioning at all was better than 1 or 2 weeks. Under long-dayconditions plants did not become as hardy, and plants acclimatedless as the duration of preconditioning was increased.The influence of a higher preconditioning temperature regime

(30 to 25 C) was examined in a parallel companion trial (study 3;data are not shown). In all treatments the hardiness was within2 C of that observed in the comparable treatments of study 2, andthe relationship of hardiness to the duration of preconditioningwas also the same.

In study 4 buds and leaves were manually removed from 60plants in the field on August 8, 1968. Any regrowth of buds orleaves was removed throughout the course of the study. Thehardiness of unaltered plants and plants with leaves and budsremoved was evaluated at five dates during the autumn (Fig. 3).Three plants were sampled in each treatment at each samplingdate. Manually defoliated plants were less hardy than theirfoliated counterparts at all sampling dates, and they were deadby the final sampling date on November 14. The presence orabsence of buds did not affect the hardiness at any of the samplingdates.A parallel companion study (study 5, data are not shown) was

conducted to examine the influence of bud and leaf removal onacclimation under long-day conditions. The experimental designwas the same except plants were exposed to 3 hr of light in themiddle of the night. The exposure to long-day treatment in thisstudy resulted in about 6 to 8 C less hardiness in foliated plantsat all sampling dates. Defoliated plants failed to acclimate (killedbelow -4 C), and they were dead at the time of the final samplingon November 14.

In study 6 leaves were manually removed from 99 plants in agrowth chamber at weekly intervals for 6 weeks and at 2-weekintervals thereafter through the 12th week. All plants wereexposed to short days and a day-night temperature regime of20 to 15 C for the first 4 weeks and 15 to 5 C thereafter for 10additional weeks. Three plants were sampled in each treatmentat each sampling date. Hardiness tests and stem length measure-ments were made at weekly intervals for 14 weeks (Fig. 4).

There was no cold acclimation before growth cessation. Leafremoval during the first 4 weeks of the study resulted in reducedstem elongation, and plants in those treatments failed to acclimate(killed below -4 C). Stems of plants in these treatments begandying back from the tips after the 12th week. Plants stoppedgrowing at about the 5th week, and those that were defoliatedanytime after that time acclimated. Acclimation was greater asthe time of defoliation was delayed. Those defoliated at the 5th

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FUCHIGAMI, WEISER, AND EVERT

ENVIRONMENTAL TREATMENTS LLOWEST SURVIVAL TEMPERATURE AFTER 17 WEEKS ( *CJPRECONDITIONING TREATMENT 1

Temperoture, 0C Duration( D-N) (weeks)

LOW TEMPERATURE (40C) I

SHORT DAYS ( 10 hours)(Duration weeks) -16 17 -18 -19 -20 -21 -22 -23 -24 -25 -26 -27

S D 1620- 15 2 15

10 H, Day 3 14

4 13

0 17

L D 1610 Hr Day 20-15 2 15

4 Hi Night 3 14Inter1upt 4 13

FIG. 2. The influence of the duration of warm (20 to 15 C) preconditioning treatments at LD and SD on subsequent cold acclimation of plantssubjected to short days and 24 C. Hardiness (lowest survival temperature) was measured after 17 weeks.

SAMPLING DATE S

FIG. 3. The influence of leaf and bud removal on the cold acclimation of C. stolonifera in the field. Leaves and buds were removed manually asindicated on August 8. Sampling dates indicate times at which hardiness determinations were made during the fall of 1968. "Dead" indicates thatdefoliated plants had died in the field before the November 14 sampling date.

week became hardy to -16 C, whereas those defoliated at the12th week acclimated to -30 C.In several other defoliation experiments, collectively referred

to as study 7 (data are not shown), plants in a growth chamberwhich provided long days and a 20 to 15-C temperature regimewere enclosed in polyethylene bags and exposed to ethylene gasover a range of concentrations from 50 to 1000,ul/liter for 12 hronce every 3 days. Treatments at the higher ethylene concentra-tions promoted senescence and abscission of older leaves to someextent. Notes were made of ethylene-induced leaf abscission every3 days, and comparable leaves were manually removed at eachdate from plants that had not been treated with ethylene. Aftermore than half of the leaves had abscised in several of the treat-ments the growth chamber was reprogrammed to provide shortdays and a temperature regime of 15 to 5 C. Periodic hardinesstests of plants defoliated manually or by ethylene treatment re-vealed no differences.

In study 8 leaves were manually removed from single branchesor whole plants in the field on July 30, as shown in Figure 5 A.In some treatments, branches were girdled by removing a 1.5-cm

band of bark tissue and scraping the exposed woody surface witha sharp scalpel. There were three plants in each treatment. Thehardiness of plants and branches in the various treatments wasevaluated on October 10.

Foliated plants and branches acclimated to -16 C. Completelydefoliated plants failed to acclimate (-4 C). Defoliated brancheson partially defoliated plants acclimated to -16 C if they wereungirdled and they failed to acclimate if the phloem was girdledbetween the defoliated and foliated parts of the plant (Fig. 5 A).

Figure 5 B shows the results of a similar growth chamber trial(study 9) in which plants were girdled and partially defoliatedbefore exposure to short days and a day-night temperature regimeof 15 to 5 C. Hardiness was tested after 37 days. As in the previousstudy defoliated branches that were girdled failed to acclimate.This was true whether the root system was isolated by girdlingfrom the defoliated branch or not.

In study 10 the leaves of plants or branches were covered withaluminum foil to exclude light as shown in Figure 6. Plants wereexposed to short days and to the standard temperature regimes(20 to 15 C for 4 weeks followed by 15 to 5 C for 8 weeks). Leaves

Day length

I -

100 Plant Physiol. Vol. 47, 1971

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Plant Physiol. Vol. 47, 1971

o 14

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z0 i8w

w

4w

2w

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-4

-6

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INDUCTION OF COLD ACCLIMATION 101

aa

b

d

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14T IM E ( WEEKS )

FIG. 4. The influence of weekly leaf removal on stem elongation and cold acclimation of C. stolonifera. Numbers (0-12) with each line indicatethe number ofweeks after the start of the experiment at which leaves were removed. Time refers to the weeks after the start of the experiment whengrowth and hardiness determinations were made. Letters (a to e) in common indicate that differences were not significant at the 0.05 level by theDuncan multiple range test.

were covered individually at either the beginning of the study orafter the first 4 weeks. Hardiness determinations were made atthe 4th, 8th, and 12th week of the study.When leaves were not covered, the plants acclimated to -8 C

by the 8th week and to -28 C by the 12th week. Plants with allthe leaves covered at the beginning of the study did not acclimate(killed below -4 C). Plants with all the leaves covered after 4weeks acclimated to a limited extent. They were hardy to -8 Cby the 8th week and to -10 C by the 12th week. Covered leavessenesced and abscised sooner than uncovered leaves. Theseresults are essentially what would be expected if leaves had beenremoved instead of covered. However, when the leaves on onebranch of a plant were covered and those on the other branchwere not, the results were not similar to those caused by defolia-

tion. Branches with covered leaves became hardier than branchesin the other treatments. By the 8th week both branches of partiallycovered plants were hardy to -14 C. This was six degrees hardierthan branches of plants with no leaves covered. At the 12th weekthe branches with covered leaves on partially covered plants werenot killed at the lowest test temperature (-32 C). Branches onthe same plants without covered leaves were less hardy (-16 and-20 C). The branches of plants on which no leaves were coveredwere hardy to -28 C.Two additional leaf covering studies (studies 11 and 12) were

conducted (data are not shown). Study 11 was the same as study10 except the leaves were covered only after 4 weeks. The resultswere similar to those of study 10. After 12 weeks the plants witheither uncovered or covered leaves were hardy to -32 and -20 C,

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FUCHIGAMI, WEISER, AND EVERT

-UNDEFOLIATED

- DEFOLIATEDI F GIRDLED

FIG. 5. The influence of girdling and leaf removal on the cold ac-climation of C. stolonifera. A: Experiment conducted in the field.Treatments were given July 30, and hardiness was tested October 10.B: Experiment conducted in a growth chamber under short-day (8-hrphotoperiod), and low temperature (15 C day and 5 C night) condi-tions. Hardiness was determined after 37 days. The illustrations on theabscissa depict the treatments and parts of plants tested for hardiness.

u0

I:3

I.-

-J

In

U,IW

Ji

0

-J

respectively. The covered leaves of branches from partially coveredplants were hardy beyond -40 C, whereas the uncovered side washardy to only -22 C.

DISCUSSION

Natural cold acclimation in C. stolonifera appears to be asequential process which proceeds most effectively when eachinductive phase is completed before proceeding to the next.Studies 1, 2, and 3 indicate that exposure to relatively high tem-peratures (20 to 15 C or 30 to 25 C) during the early phases ofshort-day induction enhances acclimation. Although it has beenreported previously that simultaneous exposure of plants to shortdays and low temperatures does not always result in optimal ac-climation (18), the cold hardiness-promoting influence of hightemperatures is not generally recognized. These results supportthe view that the early stages of acclimation are dependent uponactive metabolic processes. This viewpoint is also circumstantiallysupported by the observation of many metabolic changes whichoccur in the bark of C. stolonifera during this period (11, 12, 16-18). In a recent (unpublished) study we have also found thatinhibitors of oxidative phosphorylation and nucleic acid syn-thesis (transcription) interfere with short-day induced growthcessation in C. stolonifera explants, i.e., the inhibitors promotegrowth at short photoperiods.

Studies 2 and 3 also indicate that the duration of the warm pre-conditioning treatment is critical, and the results of study 6 sug-gest that plants (leaves) must receive a certain number of short

I 12TIME ( WEEKS )

FIG. 6. The influence of leaf covering on the controlled cold acclimation of C. stolonifera. Individual leaves on the branches indicated werecovered with aluminum foil. Illustrations on the abscissa show treatments and part of plant tested for hardiness. Time refers to the weeks from thestart of the experiment at which hardiness tests were run.

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INDUCTION OF COLD ACCLIMATION

days for acclimation. It has been proposed that many of thephotoperiod- and temperature-controlled responses of plants arerelated to the re-establishment of endogenous rhythmic patterns(4). Time is required for plants which are conditioned to a par-ticular day-night periodicity to establish new response patternswhen the environment changes. Until new patterns are wellestablished, physiological responses may be inhibited or delayed(4). This may explain why hardiness is inhibited in plants exposedto new temperature (studies 2 and 3) or photoperiod regimes forinsufficient periods of time.Growth cessation is a prerequisite to cold acclimation (study 6),

and theinduction ofgrowth cessation is probably one of the primefunctions of short days in the natural cold acclimation of plants.Conditions which promote growth, such as the warm precondi-tioning treatments combined with long photoperiods in studies2 and 3, invariably inhibit acclimation. The nature of the oblig-atory relationship between growth cessation and acclimationwhich exists in woody but not herbaceous (1) plants is probablycomplex. One simple possibility which cannot be ignored is thatgrowing plants may become depleted in the energy-producingsubstrates required for acclimation. This possibility is not un-likely under warm, long-day conditions in poorly illuminatedgrowth chambers. Removal of leaves from plants in warm en-vironments would also deplete reserves, and the poor acclimationand die-back or death of defoliated plants at the later samplingdates in studies 4, 5, and 6 may be attributable to this situation.Removal of leaves from several woody species under long-dayconditions has been reported to enhance acclimation (by removingthe source of hardiness inhibiting factors) (5, 8). This effect wasnot observed in C. stolonifera (studies 5 and 7). It is difficult tojudge whether C. stolonifera is different from the species pre-viously tested, whether the previous results are in error, or whetherthe effect was masked because defoliated plants became depletedin reserves.Although short-day induced leaves were necessary for efficient

acclimation, studies 4 and 5 indicated that buds are not involved,and studies 8 and 9 discounted the possibility of phloem-trans-ported root factors in acclimation. Results of study 7 indicatethat ethylene-induced leaf senescence and abscission have thesame inhibitory effect on acclimation as manual leaf removal.The results of the leaf removal studies (studies 4, 5, 6, 7, 8, and

9) support the concept that the short-day leaf is the source of ahardiness promoting factor(s) (3, 5, 8), and the girdling experi-ments (studies 8 and 9) clearly indicate that this promotingfactor(s) is translocated in the phloem. This finding is contraryto a previous report by Irving and Lanphear that transport is inthe xylem (8). However, the previous suggestion was based onobservations that a girdled branch exposed to long days acclimatedalmost as well as a branch on the same plant exposed to shortdays when both branches received the same prolonged exposureto low temperature (4 weeks at 4.4 C + 1 week at -1.1 C + 1day at -2.2, -3.3, -4.4, and-5.5 C). In view of the long, lowtemperature treatment, the acclimation of the girdled, long-daybranch should not have been unexpected because these same

workers (7), and others as well (5), have shown that low temper-atures can fully acclimate woody plants exposed to long days.The unusually effective acclimation of branches with aluminum

foil-covered leaves on plants where only some of the leaves werecovered (studies 10 and 11) is as interesting as it is puzzling. Sincethe foil covering caused leaves to senesce, it is possible that somesubstance produced by senescing leaves enhances acclimation.If this is true the substance must be translocated to a limitedextent only inasmuch as its hardiness-promoting effects were ap-parent only on the covered side of the plant. The results of study7 indicate that this hypothetical "senescence factor" is not ethyl-ene or a by-product of ethylene-induced senescence, and acomparison of study 12 with studies 10 and 11 indicates thatshort-day factors are also involved in this unusually effective ac-climation response.

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